A robust yet simple method to generate fluorescent amyloid nanofibers

Prajapati, Kailash Prasad; Ansari, Masihuzzaman; Yadav, Deepak Kumar; Mittal, Shikha; Anand, Bibin G.; Kar, Karunakar

doi:10.1039/d3tb01203d

Cited by 5 publications

(7 citation statements)

References 41 publications

(63 reference statements)

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“…The red puncta shown in Figure c,d confirmed the internalized Trp fibrils, and such an interpretation is strongly supported by our control experiments in which we did not see any red puncta when only Rho-B was added to the cells (Figures a,b and S4). Our previous work has confirmed that the presence of Rho-B alone at the studied concentration does not show any cytotoxic effect . Efficient internalization of Trp fibrils into SH-SY5Y cells after their treatment seems to induce cellular damage as most of the cells were found to be deformed (Figures c and f,g).…”

Section: Resultssupporting

confidence: 68%

“…Our previous work has confirmed that the presence of Rho-B alone at the studied concentration does not show any cytotoxic effect. 20 Efficient internalization of Trp fibrils into SH-SY5Y cells after their treatment seems to induce cellular damage as most of the cells were found to be deformed (Figures 2c and 1f,g). To gain insights into the mechanism of Trp fibril induced cytotoxicity, we carried out acridine orange (AO) and propidium iodide (PI) double staining assays, and the results suggested apoptosis and necrosis mediated cell death (Figure 3a,b).…”

Section: ■ Results and Discussionmentioning

confidence: 96%

“…Internalization of Trp fibrils is further supported by our control experiment on untreated cells, which did not show any blue puncta (Figure e). To further validate the efficient internalization of Trp fibrils, we visualized the RhoB-incorporated Trp aggregates using a coaggregation reaction protocol . RhoB incorporated Trp fibrils were subjected to washing and resuspension steps after centrifugation prior to cellular treatment.…”

Section: Resultsmentioning

confidence: 99%

“…To further validate the efficient internalization of Trp fibrils, we visualized the RhoB-incorporated Trp aggregates using a coaggregation reaction protocol. 20 RhoB incorporated Trp fibrils were subjected to washing and resuspension steps after centrifugation prior to cellular treatment. Addition of RhoBincorporated Trp fibrils allowed us to detect their cellular internalization (Figure 2c,d).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The morphology of the insulin fibrils obtained from the Trp-seeded aggregation reactions showed typical fibrillar structures that match well with previously reported morphology of insulin fibrils (Figure 5e). 34−38 To explore any possible interactions between higher order structures of insulin and Trp, we conducted FRET-based experiments following established protocols 20,29 using fluorescein-incorporated insulin fibrils (Figure 5f) and Rho-B incorporated Trp fibrils (Figure 5g). FRET signal comprising emission from both Rho-B and fluorescein was detected when the sample containing a mixture of these two fibrils was excited at 490 nm (specific for fluorescein, as donor) (Figure 5h).…”

Section: Seeding Effect Of Differently Aged Trp Aggregatesmentioning

confidence: 99%

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Initiation of Brain Extract Fibrillation and Effective Cellular Internalization of Tryptophan Fibrils Unveils Its Neurotoxicity Risk

Prajapati,

Ansari,

Mittal

et al. 2023

ACS Chem. Neurosci.

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Recent discoveries on the self-assembly of aromatic amino acids into amyloid-like neurotoxic nanostructures have initiated a quest to decode the molecular mechanisms for the initiation of neurodegeneration. Moreover, the multicomponent nature of the amyloid deposits still questions the existing and well-defined amyloid cascade hypothesis. Hence, deciphering the neurotoxicity of amyloid-like nanostructures of aromatic amino acids becomes crucial for understanding the etiology of amyloidogenesis. Here, we demonstrate the cellular internalization and consequential damaging effects of self-assembled amyloid-like tryptophan nanostructures on human neuroblastoma cells. The cell-damaging potential of tryptophan nanostructure seems to be facilitated via ROS generation, necrosis and apoptosis mediated cell death. Further, tryptophan nanostructures were found to be seeding competent conformers, which triggered aggressive aggregation of brain extract components. The early stage intermediate nanostructures possess a higher cross-seeding efficacy than the seeding potential of the matured tryptophan fibrils. In addition to the cell-damaging and cross-seeding effects, tryptophan fibrils were found to catalyze oxidation of neuromodulator dopamine. These findings add more insights into the specific role of tryptophan self-assembly during the pathogenesis of hypertryptophanemia and other amyloid-associated neurodegenerative complications.

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Section: Resultssupporting

confidence: 68%

Section: ■ Results and Discussionmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Seeding Effect Of Differently Aged Trp Aggregatesmentioning

confidence: 99%

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Initiation of Brain Extract Fibrillation and Effective Cellular Internalization of Tryptophan Fibrils Unveils Its Neurotoxicity Risk

Prajapati,

Ansari,

Mittal

et al. 2023

ACS Chem. Neurosci.

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Rapid Coaggregation of Proteins Without Sequence Similarity: Possible Role of Conformational Complementarity

Prajapati,

Ansari,

Mittal

et al. 2024

Biochemistry

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Despite extensive research on the sequencedetermined self-assembly of both pathogenic and nonpathogenic proteins, the question of how the sequence identity would influence the coassembly or cross-seeding of diverse proteins without distinct sequence similarity remains largely unanswered. Here, we demonstrate that the rapid coaggregation of proteins with negligible sequence similarity is fundamentally governed by preferred heteromeric interactions between their partially unfolded states via the gain of additional charge complementarity and hydrophobic interactions. The partial loss of intramolecular interactions and concurrent gain of non-native intrinsically disordered regions with sticky groups become crucial for both aggressive heteromeric primary nucleation and secondary nucleation events. The results signify the direct relevance of sequenceindependent conformational cross-talk between diverse proteins to the foundational events required for the growth of biological multiprotein amyloid deposits.

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Pleiotropic Nanostructures Built from l-Histidine Show Biologically Relevant Multicatalytic Activities

Prajapati,

Mittal,

Ansari

et al. 2024

ACS Appl. Mater. Interfaces

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The essential amino acid histidine plays a central role in the manifestation of several metabolic processes, including protein synthesis, enzyme-catalysis, and key biomolecular interactions. However, excess accumulation of histidine causes histidinemia, which shows brain-related medical complications, and the molecular mechanism of such histidine-linked complications is largely unknown. Here, we show that histidine undergoes a self-assembly process, leading to the formation of amyloid-like cytotoxic and catalytically active nanofibers. The kinetics of histidine self-assembly was favored in the presence of Mg(II) and Co(II) ions. Molecular dynamics data showed that preferential noncovalent interactions dominated by H-bonds between histidine molecules facilitate the formation of histidine nanofibers. The histidine nanofibers induced amyloid cross-seeding reactions in several proteins and peptides including pathogenic Aβ 1−42 and brain extract components. Further, the histidine nanofibers exhibited oxidase activity and enhanced the oxidation of neurotransmitters. Cell-based studies confirmed the cellular internalization of histidine nanofibers in SH-SY5Y cells and subsequent cytotoxic effects through necrosis and apoptosis-mediated cell death. Since several complications including behavioral abnormality, developmental delay, and neurological disabilities are directly linked to abnormal accumulation of histidine, our findings provide a foundational understanding of the mechanism of histidine-related complications. Further, the ability of histidine nanofibers to catalyze amyloid seeding and oxidation reactions is equally important for both biological and materials science research.

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A robust yet simple method to generate fluorescent amyloid nanofibers

Abstract: Covalent tagging of fluorophores is central to the mechanistic understanding of important biological processes including protein-protein interaction and protein aggregation. Hence, studies on fluorophore-tagged peptides help in elucidating the molecular...

Cited by 5 publications

References 41 publications

Initiation of Brain Extract Fibrillation and Effective Cellular Internalization of Tryptophan Fibrils Unveils Its Neurotoxicity Risk

Initiation of Brain Extract Fibrillation and Effective Cellular Internalization of Tryptophan Fibrils Unveils Its Neurotoxicity Risk

Rapid Coaggregation of Proteins Without Sequence Similarity: Possible Role of Conformational Complementarity

Pleiotropic Nanostructures Built from l-Histidine Show Biologically Relevant Multicatalytic Activities

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